Myo-inositol analogs and method for their use

ABSTRACT

The present invention relates to a method for inhibiting the phosphatidylinositol cycle in a cell by contacting the cell with certain myo-inositol analogs. Such myo-inositol analogs can also be utilized to treat phosphatidylinositol cycle-dependent conditions. The present invention also provides novel myo-inositol analogs.

TABLE OF CONTENTS

1. Technical Field

2. Background of the Invention

3. Summary of the Invention

4. Detailed Description of the Invention

4.1 The Method of the Invention

4.1.1 In-Vitro Method

4.1.2 In-Vivo method

4.2 Compounds of the Present Invention

4.3 Mode of Administration and Pharmaceutical Compositions

5. Examples

5.1 Synthesis of Compounds of Formula I

5.2 Biological Data

1. TECHNICAL FIELD

The present invention relates to a method for inhibiting thephosphatidylinositol cycle in a cell by contacting the cell with certainmyo-inositol analogs. Such myo-inositol analogs can also be utilized totreat phosphatidylinositol cycle-dependent conditions. The presentinvention also provides novel myo-inositol analogs.

2. BACKGROUND OF THE INVENTION

For a cell to survive, it must be able to respond rapidly to changes inits environment. Furthermore, for cells to reproduce and carry out otherco-operative functions, they must be able to communicate efficientlywith each other. Cells most frequently adapt to their environment andcommunicate with one another by means of chemical signals. An importantfeature of these signaling mechanisms is that in almost all cases a cellis able to detect a chemical signal without it being necessary for thechemical messenger itself to enter the cell. This permits the cell tomaintain tight control of its internal milieu, thereby permitting thecell to respond to its environment without being destroyed by it.

These sensing functions are carried out by a variety of receptors, whichare dispersed on the outer surface of the cell and function as molecularantennae. These receptors detect an incoming messenger and activate asignal pathway that ultimately regulates a cellular process such assecretion, contraction, metabolism or growth. The major barrier to theflow of information is the cell's cellular plasma membrane, wheretransduction mechanisms translate external signals into internalsignals, which are then carried throughout the interior of the cell by"second messengers."

In molecular terms, the process depends on a series of proteins withinthe cellular plasma membrane, each of which transmits information byinducing a conformational change--an alteration in shape and thereforein function--in the protein next in line. At some point the informationis assigned to small molecules or even to ions within the cell'scytoplasm, which serve as the above-mentioned second messengers, whosediffusion enables a signal to propagate rapidly throughout the cell.

The number of second messengers appears at present to be surprisinglysmall. To put it another way, the internal signal pathways in cells areremarkably universal, and have been phylogenetically preserved overmillions of years of evolution. Yet the known messengers are capable ofregulating a vast variety of physiological and biochemical processes.The discovery of the identity of particular second-messenger substancesis proving, therefore, to be of fundamental importance for understandinghow cellular growth and function are regulated.

Several major signal pathways are now known, but two seem to be ofprimary importance. One employs cyclic nucleotides as second-messengers.These cyclic nucleotides activate a number of proteins inside the cell,which then cause a specific cellular response. The other major pathwayemploys a combination of second messengers that includes calcium ions aswell as two substances whose origin is remarkable: inositol 1, 4, 5tri-phosphate (IP₃) and diacylglycerol (DG). These compounds arecannibalized from the plasma membrane itself, by enzymes which areactivated by specific cellular membrane receptors. However, it should benoted that inositol in its non-phosphorylated form first enters anorganism through the organism's diet, but can then be recycled asdescribed hereinbelow.

IP₃ is formed by the following scheme. A receptor molecule on thesurface of the cellular plasma membrane transmits information throughthe cellular plasma membrane and into the cell by means of a family of Gproteins, which are cellular plasma membrane proteins that cannot beactive unless they bind to guanosine triphosphate (GTP). The G proteinsactivate the so-called "amplifier" enzyme phospholipase C, which is onthe inner surface of the cellular plasma membrane. Phospholipase Ccleaves the cellular plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP₂) into DG and IP₃. IP₃ is a water-soluble molecule,and therefore, upon being released from the inner surface of thecellular plasma membrane, it rapidly diffuses into cytoplasm. IP₃ thenreleases calcium from internal compartments, which store highconcentrations of calcium. The calcium released by IP₃ in turn activatesa large number of intracellular enzymes that orchestrate a complex setof responses that allow the cell to adapt to the original signaltriggering the receptor that caused the release of IP₃.

Quite fascinatingly, DG and IP₃ are recycled. DG is recycled by a seriesof chemical reactions which constitute one component of the lipid cycle.IP₃ is recycled by a series of reactions known as thephosphatidylinositol cycle. The two cycles converge at the point wheninositol is chemically linked to DG. The DG-bound inositol isphosphorylated in a series of steps which ultimately results in thereformation of phosphatidylinositol diphosphate.

In the first portion of the lipid cycle, DG is converted to phosphatidicacid, which in turn is converted to

cytidine diphosphate deglyceride (CDP--DG), while in the first portionof the phosphatidylinositol cycle, IP₃ is dephosphorylated to ultimatelyform myo-inositol. [Note: "myo" refers to the stereochemistry of theinositol molecules. Since all known inositol second messengers use themyo-configuration of inositol, the term "inositol" will herein beunderstood to refer to myo-inositol.] It is believed that suchdephosphorylation occurs stepwise; IP₃ is converted to an inositolbearing only two phosphate groups (IP₂), followed by the loss of anadditional phosphate, resulting in IP₁, which is then dephosphorylatedto myo-inositol. Also, it has been shown that IP₃ can also undergo anadditional phosphorylation, thereby being converted to inositol 1, 3, 4,5 tetra-phosphate (IP₄). This molecule is subsequently metabolized bysuccessive removal of phosphate groups, as described above. It isbelieved that a phosphatase enzyme catalyses each step of this process.

The lipid cycle and phosphatidylinositol cycle merge by the myo-inositolreacting with the CDP--DG to form phosphatidylinositol (PI). PI isphosphorylated to ultimately form PIP₂. It is believed that suchphosphorylation occurs stepwise; PI is converted to phosphatidylmyo-inositol 4-phosphate (PIP), which is converted to PIP_(2;) the finalstep of both cycles. It is believed that a kinase enzyme catalyses eachstep of this process.

For an excellent review of IP₃, its role as a second messenger and thephosphatidylinositol cycle see Berridge, M., et al. InositolTriphosphate, a Novel Second Messenger in Cellular Signal Transduction,Nature, 312, 315-321 (1984) and Berridge, M., The Molecular Basis ofCommunication Within the Cell, Scientific American, 142-152 (October1985), and James W. Putney, Jr. (Ed.), Phosphoinositides and ReceptorMechanisms, Alan R. Liss, Inc., New York, N.Y. 1986.

3. SUMMARY OF THE INVENTION

The present invention relates to a method for inhibiting thephosphatidylinositol cycle in a cell which comprises contacting saidcell with a compound represented by the formula I: ##STR1## wherein:

R¹, R², R³, R⁴, R⁵ and R⁶ is each independently selected from the groupconsisting of OH, F, N₃, NH₂, SH, SeH, Cl, Br, I and CN; with theproviso:

(i) four or five of said R¹, R², R³, R⁴, R⁵ and R⁶ are OH; and

(ii) if said R⁴, R⁵ or R⁶ is F, Cl, Br or I then four of said R¹, R²,R³, R⁴, R⁵ and R⁶ are OH.

The present invention also provides a method for treatingphosphatidylinositol cycle-dependent conditions.

The present invention also provides compounds represented by the formulaII: ##STR2## wherein:

R¹, R², R³, R⁴, R⁵ and R⁶ is each independently selected from the groupconsisting OH, F, N₃, NH₂, SH, SeH, Cl, Br, I and CN; with the proviso:

(i) four or five of said R¹, R², R³, R⁴, R⁵ and R⁶ are OH;

(ii) if one of said R¹, R², R³, R⁴, R⁵ and R⁶ is N₃, then one of saidR¹, R², R³, R⁴, R⁵ and R⁶ is selected from the group consisting of F,NH₂, SH, SeH, Cl, Br, I and CN;

(iii) if one of said R¹, R², R³, R⁴, R⁵ and R⁶ is NH₂, then one of saidR¹, R², R³, R⁴, R⁵ and R⁶ is selected from the group consisting of F,N₃, SH, SeH, Cl, Br, I and CN; and

(iv) if said R⁴, R⁵ or R⁶ is F, Cl, Br or I then four of said R¹, R²,R³, R⁴, R⁵ and R⁶ are OH.

4. DETAILED DESCRIPTION OF THE INVENTION 4.1 The Method of the Invention

The present invention relates to a method for inhibiting thephosphatidylinositol cycle in a cell which comprises contacting saidcell with a compound represented by formula I: ##STR3## wherein:

R¹, R², R³, R⁴, R⁵ and R⁶ is each independently selected from the groupconsisting OH, F, N₃, NH₂, SH, SeH, Cl, Br, I and CN; with the proviso:

(i) four or five of said R¹, R², R³, R⁴, R⁵ and R⁶ are OH; and

(ii) if said R⁴, R⁵ or R⁶ is F, Cl, Br or I then four of said R¹, R²,R³, R⁴, R⁵ and R⁶ are OH.

It is believed that the compounds of formula I, which are analogs ofmyo-inositol, are effective for inhibiting the phosphatidylinositolcycle. Without being bound by theory, it is believed that the compoundsof formula I inhibit the phosphatidylinositol cycle by two mechanisms.Firstly, the compounds of formula I compete with myo-inositol for beingtransported into the cell and, therefore, less myo-inositol is availablein the cell to be utilized in the phosphatidylinositol cycle. Secondly,the phosphatase enzymes and kinase enzymes of the phosphatidylinositolcycle cannot dephosphorylate and phosphorylate, respectively, thepositions of the compounds of formula I that are substituted with F, N₃,NH₂, SH, SeH, Cl, Br, I or CN, thereby inhibiting such enzymes frombeing utilized efficiently in the phosphatidylinositol cycle, thusinhibiting such cycle.

4.1.1 In-Vitro Method

This method can be carried out either in-vitro or in-vivo. In-vitro, thecompounds of formula I can be utilized as pharmacological tools forstudying cellular response to, for example, hormones, neuropeptides,neurotransmitters or synthetic drugs. Such compounds can also beutilized to study the involvement of the phosphatidylinositol cycle incellular growth or cellular differentiation.

The compounds of formula I can also be radiolabeled, e.g., tritiated,which renders it easier to detect the presence of such compounds incells. Also, radiolabeling can be utilized to study what proportion ofthe compounds of formula I enters the cells.

4.1.2. In-Vivo Method

The method can also be utilized in-vivo which comprises a method forinhibiting the phosphatidylinositol cycle in a mammal, including humans,which comprises administering to said mammal an phosphatidylinositolcycle inhibiting amount of a compound of formula I.

The present invention also covers the use of the compounds of formula Ito treat phosphatidylinositol cycle-dependent conditions in mammals,including humans. This aspect of the present invention comprises amethod for treating phosphatidylinositol cycle-dependent conditions in amammal which comprises administering to said mammal anphosphatidylinositol cycle inhibiting amount of a compound of formula I.

Inositol phosphate cycle-dependent conditions include abnormal cellulargrowth as found in neoplastic conditions, as well as biochemicalprocesses relevant to arthritis, pain, inflammation, and plateletaggregation. See Y. Nishizuka, Science, 225, 1365-1370 (1984); S. K.Fisher and B. W. Agranoff, J. Neurochem., 48, 999-1017 (1987); Y.Sugimoto and R. I. Erikson, Molecular and Cellular Biology,5 3194-3198(1985); K. Fukami, K. Matsuoka, O. Nakanishi, A. Yamakawa, S. Kawai, andT. Takenawa, Proc. Natl. Acad. Sci., U.S.A., 85, 9057-9061 (1988); M.Whitman, L. Fleischman, S. B. Chahwala, L. Cantley, and P. Rosoff, inPhosphoinositides and Receptor Mechanisms, 195-217, A. R. Liss, Inc.1986.

4.2. Compounds of the Present Invention

The present invention also provides compounds represented by the formulaII: ##STR4## wherein:

R¹, R², R³, R⁴, R⁵ and R⁶ is each independently selected from the groupconsisting of OH, F, N₃, NH₂, SH, SeH, Cl, Br, I and CN; with theproviso:

(i) four or five of said R¹, R², R³, R⁴, R⁵ and R⁶ are OH;

(ii) if one of said R¹, R², R³, R⁴, R⁵ and R⁶ is N₃, then one of saidR¹, R², R³, R⁴, R⁵ and R⁶ is selected from the group consisting of F,NH₂, SH, SeH, Cl, Br, I and CN;

(iii) if one of said R¹, R², R³, R⁴, R⁵ and R⁶ is NH₂, then one of saidR¹, R², R³, R⁴, R⁵ and R⁶ is selected from the group consisting of F,N₃, SH, SeH, Cl, Br, I and CN; and

(iv) if said R⁴, R⁵ or R⁶ is F, Cl, Br or I then four of said R¹, R²,R³, R⁴, R⁵ and R⁶ are OH.

The preferred compounds of formula II are wherein five of said R¹, R²,R³, R⁴, R⁵ and R⁶ are OH, i.e. mono-substituted compounds, and one ofsaid R¹, R², R³, R⁴, R⁵ and R⁶, and more preferably said R¹, is selectedfrom the group consisting of F, SH, SeH, Cl, Br, I and CN. However, dueto that the 3-position substitution is very easy to synthesize, anotherpreferred embodiment of the present invention is wherein said R¹, R²,R⁴, R⁵ and R⁶ is OH and R³ is selected from the group consisting of F,SH, SeH, Cl, Br, I and CN.

Of the non-OH substituents, F and SH are preferred, with F being mostpreferred.

A less preferred embodiment of formula II is wherein four of said R¹,R², R³, R⁴, R⁵ and R⁶ are OH and two of said R¹, R², R³, R⁴, R⁵ and R⁶are selected from the group consisting of F, N₃, NH₂, SH, SeH, Cl, Br, Iand CN, i.e. disubstituted compounds. Of such disubstituted compounds,it is preferred that two of said R¹, R⁴ and R⁵, yet more preferably saidR⁴ and R⁵ be independently selected from the group consisting of F, N₃,NH₂, SH, SeH, Cl, Br, I and CN.

Of the non-OH substituents of the disubstituted compounds, preferred areF and SH; F and NH₂ ; NH₂ and SH; SH and SH; and F and F; with F and Fbeing most preferred.

The preferred compounds of formula II are also the preferred compoundsfor use in the methods of the present invention.

4.3. Mode of Administration and Pharmaceutical Compositions

When the compounds of formula I are utilized in vivo, such compounds canbe administered orally, topically, parentally, by inhalation spray orrectally in dosage unit formulations containing conventional non-toxicpharmaceutically acceptable carriers.

Accordingly, the present invention also provides pharmaceuticalcompositions comprising the compounds of formula II with apharmaceutically acceptable carrier.

The term parenteral as used herein includes subcutaneous injections,intravenous, intramuscular, intrasternal injection or infusiontechniques In addition to the treatment of mammals, such as mice, rats,horses, dogs, cats, etc., the compounds of the invention are effectivein the treatment of humans.

The pharmaceutical compositions containing the active ingredient may bein a form suitable for oral use, for example, as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard or soft capsules, or syrups or elixirs Compositionsintended for oral use may be prepared according to any method known tothe art for the manufacture of pharmaceutical compositions and suchcompositions may contain one or more agents selected from the groupconsisting of sweetening agents, flavoring agents, coloring agents andpreserving agents in order to provide pharmaceutically elegant andpalatable preparation.

Formulations for oral use include tablets which contain the activeingredient in admixture with non-toxic pharmaceutically acceptableexcipients. These excipients may be, for example, inert diluents, suchas calcium carbonate, sodium carbonate, lactose, calcium phosphate orsodium phosphate, granulating and disintegrating agents, for example,maize starch, or alginic acid; binding agents, for example, starch,gelatin or acacia, and lubricating agents, for example, magnesiumstearate, stearic acid or talc. The tables may be uncoated or they maybe coated by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl disterate may be employed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example, peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions usually contain the active materials in admixturewith appropriate excipients Such excipients are suspending agents, forexample, sodium carboxymethylcellulose, methylcellulose,hydroxypropymethylcellulose, sodium alinate, polyvinylpryrolidone, gumtragacanth and gum acacia; dispersing or wetting agents which may be anaturally-occurring phosphatide, for example, lecithin; a condensationproduct of an alkylene oxide with a fatty oxide, for example,polyoxyethylene stearate; a condensation product of ethylene oxide witha long chain aliphatic alcohol, for example,heptadecaethyleneoxycetanol; a condensation product of ethylene oxidewith a partial ester derived from fatty acids and a hexitol such aspolyoxyethylene sorbitol monooleate; or a condensation product ofethylene oxide with a partial ester derived from fatty acids and hexitolanhydrides, for example, polyoxyethylene sorbitan monooleate. Theaqueous suspensions may also contain one or more preservatives, forexample, ethyl, n-propyl, or p-hydroxybenzoate; one or more coloringagents; one or more flavoring agents; and one or more sweetening agentssuch as sucrose or saccharin.

Oily suspension may be formulated by suspending the active ingredientsin a vegetable oil, for example, arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspension may contain a thickening agent, for example, beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of anantioxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending and one or morepreservatives. Suitable dispersing or wetting agents and suspendingagents are exemplified by those already mentioned above. Additionalexcipients, for example, sweetening, flavoring and coloring agents, mayalso be present.

The pharmaceutical compositions of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil, forexample, olive oil or arachis oils, or a mineral oil, for example,liquid paraffin or mixtures of those. Suitable emulsifying agents may benaturally-occurring gums, for example, gum acacia or gum tragacanth,naturally-occurring phosphatides, for example, soybean lecithin; andesters including partial esters derived from fatty acids and hexitolanhydrides, for example, sorbitan mono-oleate, and condensation productsof said partial esters with ethylene oxide, for example, polyoxyethylenesorbitan mono-oleate. The emulsions may also contain sweetening andflavoring agents.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, sorbitol or sucrose. Such formulations may also contain ademulcent, a preservative and flavoring and coloring agents. Thepharmaceutical compositions may be in the form of a sterile injectableaqueous or oleaginous suspension. This suspension may be formulatedaccording to the known art using those suitable dispersing or wettingagents and suspending agents which have been mentioned above. Thesterile injectable preparation may be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent.Among the acceptable vehicles and solvents that may be employed arewater, 1,3-butanediol, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile fixed oils are conventionally employed asa solvent or suspending medium. For this purpose any bland fixed oil maybe employed including synthetic mono- or diglycerides. Fatty acids suchas oleic acid also find use in the preparation of injectables.

The compounds of formula I can also be administered in the form ofsuppositories for rectal administration of the drug. These compositionscan be prepared by mixing the drug with a suitable non-irritatingexcipient which is solid at ordinary temperatures but liquid at therectal temperature and will therefore melt in the rectum to release thedrug, for example, cocoa butter and polyethylene glycols.

When the compounds of formula I are utilized in vivo, dosage levels onthe order of from about 0.2 mg to about 300 mg, preferably from about 10mg to about 100 mg, per kilogram of body weight per day are useful.

5. EXAMPLES 5.1. Synthesis of Compounds of Formula I EXAMPLE I Synthesisof 3-deoxy-3-fluoro-myo-inositol and 1-deoxy-1-fluoro-myo-inositol

The following synthesis is with reference to the scheme set forth inScheme 1 hereinbelow. (The number after the compounds referred to inthis scheme refer to the compounds designated by the same number inScheme 1. Also, some compounds referred to in the scheme are onlyreferred to by the number, which also refers to the compound in Scheme Idesignated by such number. The subsequent examples are written in thesame manner.)

Preparation of Monocyclohexylidene Ketal 2

A 2-L flask equipped with a mechanical stirrer, a Dean-Stark trap and areflux condenser was charged with myo-inositol (50 g), cyclohexanone(500 mL), and anhydrous benzene (130 mL). The mixture was stirred andrefluxed until no more water separated. ρ-Toluensulphonic acidmonohydrate (p-TsOH) (0.5 g) was added and the mixture was refluxeduntil the separation of water was complete and almost all themyo-inositol had dissolved (about 4 hr.). The mixture was cooled anddecanted from the remaining solid, which was washed with 50 mL ofbenzene. More benzene (200 mL), light petroleum (b.p. 60°-80°) (250 mL),and ethanol (50 mL) were added in this order to the solution. Thesolution was kept at 0° C. overnight. Triethylamine (1 mL) was added andthe solid was filtered and washed with benzene The crude product waspurified by extraction with boiling ethanol (1000 mL) containingtriethylamine (1 mL). On cooling, the product (ca 54 g. 74%)crystallized.

Preparation of Diol 3

A mixture of the monocyclohexylidine ketal 2 (36.8 g, 0.142 mol), benzylchloride (407 g, 3.21 mol), and potassium hydroxide (220 g, 3.93 mol)was stirrred with a mechanical stirrer at 90°-100° C. for 20 hours. Thecooled mixture was diluted with benzene (400 mL) and washed with water(4×150 mL). The benzene solution was concentrated on a rotaryevaporator, and the residue was heated in glacial acetic acid (800 mL)and water (160 mL) at 90°-l00° C. for 4 hours. The solution wasconcentrated on a rotary evaporator at 55° C. and the residue waspurified by column chromatography (30% ethyl acetate in hexanes than 40%ethyl acetate in hexanes); yield 36 g (42%) of 3.

Preparation of Monobenzoyl Ester 4

Benzoyl chloride (1.1 mol. equiv.) was added dropwise to a solution ofthe diol 3 (1 g) in pyridine (50 mL). The solution was stirred at roomtemperature for 4 hours. The pyridine was evaporated and the residue wastaken up in ethyl acetate and washed with water. After drying andconcentration, the crude product was purified by flash chromatography(40% ethyl acetate in hexanes); yield 80%.

Fluorination of Monobenzoyl Ester 4

To a solution of the monobenzoyl ester 4 in toluene at 80° C. was addeddropwise diethylamino-sulfur trifluoride (DAST) (2 mol equiv.). Thesolution was kept at 80° C. for 1 hour. After cooling down to roomtemperature, the solution was diluted with benzene and washed withbrine. The solution was dried over MgSO₄, filtered, and concentrated.Flash chromatography (20% ethyl acetate in hexanes) gave the pureproduct 5 in 76% yield.

Preparation of Racemic Equatorial Alcohol 6

Methanol was added to a mixture of the fluorinated monobenzoyl ester 5(0.37 g, 0.58 nmol) in 5% NaOH-THF (1:1, 40 mL) until the mixture becamehomogeneous. The solution was stirred at room temperature for 1 hour.Methanol and THF were removed by a rotary evaporator. The aqueous layerwas extracted with ethyl acetate. The extracts were washed with brine,dried, and concentrated. Flash chromatography (20% ethyl acetate inhexanes) gave 0.23 g of racemic alcohol 6 (73%).

Preparation of (-) and (+) Diastereomeric Camphanic Esters [(-) and (+)7, respectively]

(S)-(-)-camphanic acid chloride (1.11 g, 2 mol equiv.) was added inseveral portions quickly to a solution of racemic alcohol 6 (1.38 g,2.55 mmol), triethylamine (1.76 mL, 5 mol equiv.), and a catalyticamount of 4-dimethylaminopyridine (DMAP) in dry dichloromethane (50 mL)at room temperature. The solution was stirred at room temperature for 2hours. The solution was then diluted with dichloromethane, washed withsaturated NH₄ Cl, dried and concentrated. Flash chromatography (FC) (20%ethyl acetate in hexanes, 100 g of 230-400 mesh silica gel per gram ofcrude product) separated the two diastereomeric camphanic esters withthe less polar one being the (-) camphanic ester 7 (3-fluoro-) and themore polar one being the (+) camphanic ester 7 (1-fluoro-). The combinedyield was approximately 90%.

Hydrolysis of the (-)-Camphanic Ester 7

Methanol was added to a mixture of the (-)-camphanic ester 7 (332 mg,0.460 nmol) in 1:1 5% NaOH--THF (60 mL) at room temperature until themixture became homogeneous. Stirring was continued at room temperaturefor 2 hours. MeOH and THF were evaporated on a rotavap. The resultingaqueous mixture was extracted with AcOEt. The organic phase was washedwith brine, dried and concentrated Flash chromatography (20% ethylacetate in hexanes) gave the (+)-alcohol 8 (237 mg. 95%).

Oxidation of (+)-Alcohol 8

To a solution of oxalyl chloride (3 mol equiv.) in dry CH₂ Cl₂ at -78°C. under N₂ was added dimethylsulfoxide (DMSO) (7 mol equiv.). Afterstirring at -78° C. for 15 minutes, a solution of the (+)-alcohol 8 indichloromethane (1 g in 5 mL) was introduced dropwise through a syringe.Stirring was continued at -78° C. for 1 hour. Triethylamine (10 molequiv.) was added and the resulting solution was allowed to warm upslowly to room temperature. Water was added to the solution and volatileorganics were evaporated on a rotary evaporator. The resulting aqueousmixture was extracted with AcOEt. The organic phase was washed withwater (3 times) and brine, dried and concentrated. The ketone 9 (ca.80%) was used directly for the next step.

Reduction of Ketone 9

To a solution of the ketone 9 (0.826 g, 1.532 mmol) in dry THF (50 mL)at -78° C. under N₂ was added dropwise L-Selectride (2.30 mL of a 1Msolution in THF, 1.5 mol equiv.). The solution was warmed slowly to roomtemperature. Saturated NH₄ Cl was added to quench the excess reagent.THF was evaporated and the aqueous mixture was extracted with AcOEt. Theorganic layer was washed with brine, dried and concentrated. Flashchromatography (20% ethyl acetate in hexanes) gave the (+)-axial alcohol10 (0.669 g, 81%).

Hydrogenolysis of the (+)-Axial Alcohol 10

A mixture of the (+)-axial alcohol 10 (177 mg.), several drops of 5%HCl, and 10% Pd/C (equal weight with the axial alcohol(+)-10) in EtOH(30 mL) was stirred under an atmosphere of H₂ at room temperatureovernight. The catalyst was filtered, and the filtrate was concentratedfirst on a rotary evaporator and then by high vacuum to yield (greaterthan 95% yield) 3-deoxy-3-fluoro-myo-inositol. Recrystallization frommethanol gave crystalline 3-deoxy-3-fluoro-myo-inositol.

The same chemical synthesis can be utilized to prepare1-deoxy-1-fluoro-myo-inositol, which is the enantiomer of3-deoxy-3-fluoro-myo-inositol, except that (-)8 compound rather than the(+)8 compound is utilized. ##STR5##

The spectral data for the compounds of this example was as follows:

2: ¹ H NMR (D₂ O): δ 4.45 (t,J=4.4 Hz, 1 H), 4.02 (dd, J-7.7, 5.1 Hz, 1H), 3.82 (dd, J=9.7, 4.1 Hz, 1 H), 3.52-3.65 (m, 2 H), 3.23 (t, J=10.0Hz, 1 H), 1.34-1.73 (m, 10 H).

3: R_(f) (40% ethyl acetate in hexanes)=0.22; ¹ H NMR: δ 7.23-7:30 (m,20 H), 4.69-4.94 (m, 8 H}, 4.17 (br s, 1 H), 3.96 (t, J=9.4 Hz, 1 H),3.82 (t, J=9.4 Hz, 1 H), 3.42-3.49 (m, 3 H), 2.53 (br s, --OH), 2.44 (brd, J =3.8 Hz, --OH).

4: R_(f) (40% ethyl acetate in hexanes)=0.67; ¹ H NMR: δ 7.13-8.18 (m,25 H), 5.11 (br d, J=10.2 Hz, 1 H), 4.70-4.94 (m, 8 H), 4.42 (br s, 1H), 4.24 (t, J=9.4 Hz, 1 H), 4.02 (t, J=9.4 Hz, 1 H), 3.58-3.64 (m, 2H), 2.54 (br s, --OH).

5: R_(f) (20% ethyl acetate in hexanes)=0.37; ¹ H NMR: δ 7.05-8.04 (m,25 H), 5.53 (dt, J=12.1, 9.7 Hz, H), 4.62-4.93 (m, 8 H), 4.61 (dt, J=51,9.5 Hz, 1 H), 3.80 (dt, J=12.7, 9.2 Hz, 1 H), 3.57-3.75 (m, 3 H).

6: R_(f) (20% ethyl acetate in hexanes)=0.15; ¹ H NMR: δ 7.24-7.37 (m,20 H), 4.74-4.95 (m, 8 H) 4.42 (dt, J=51, 8.9 Hz, 1 H), 3.51-3.78 (m, 4H), 3.39 (t, J=9.0 Hz, 1 H), 2.49 (d, J=2 Hz, --OH).

(-)-7: white solid, mp=136°-138° C.;

R_(f) (20% ethyl acetate in hexanes)=0.22;

IR: 3063, 3031, 2967, 2933, 2878, 1791, 1741, 1496, 1453, 1397, 1360,1337, 1315, 1271, 1212, 1163, 1141, 1098, 1061, 1028, 993, 930, 736, 698cm⁻¹ ;

¹ H NMR: δ 7.22-7.33 (m, 20 H), 5.34-5.43 (m, 1 H), 4.68-4.90 (m, 8 H),4.54 (dt, J=51, 9.3 Hz, 1 H), 3.52-3.79 (m, 4 H), 2.36-2.45 (m, 1 H),1.73-1.94 (m, 2 H), 1.56-1.67 (m, 1 H), 1.11 (s, 3 H), 1.04 (s, 3 H),0.99 (s, 3 H);

Mass spectrum (m/z): 631 (M⁺ -91), 525, 433, 327, 299, 197, 181, 91, 55,exact mass calcd for C₃₇ H₄₀ FO₈ 631.2707, found 631.2705; Anal. Calcdfor C₄₄ H₄₇ FO₈ : C,73.11; H, 6.55; F, 2.63, found: C, 72.94; H, 6.33;F, 2.49;

[α]_(D) ²⁴ =7.6° (c 5.4, CHCl₃).

(+)-7: glassy solid;

R_(f) (20% ethyl acetate in hexanes)=0.16;

IR: 3062, 3031, 2965, 2932, 2879, 1792, 1761, 1497, 1453, 1397, 1360,1333, 1310, 1264, 1214, 1142, 1061, 1028, 933, 736, 698 cm⁻¹ ;

¹ H NMR: δ 7.18-7.32 (m, 20 H), 5.42 (dt, J=11, 9.7 Hz, 1 H), 4.64-4.91(m, 8 H), 4.51 (dt, J-51, 9.4 Hz, 1 H), 3.52-3.80 (m, 4 H), 2.30-2.40(m, 1 H), 1.85-2.00 (m, 2 H), 1.63-1.72 (m, 1 H), 1.09 (s, 3 H), 1.05(s, 3 H), 0.88 (s, 3. H);

Mass spectrum (m/z): 631 (M⁺ --91), 525, 433, 327, 299, 197, 181, 91;exact mass calcd for C₃₇ H₄₀ FO₈ 631.2707, found 631.2705; Anal. Calcdfor C₄₄ H₄₇ FO₈ : C, 73.11; H, 6.55; F, 2.63, found: C, 72.77; H, 6.51;F, 2.49;

[α]_(D) ²⁴ =+0.91° (c 2.3, CHCl₃).

(+)-8 (derived by hydrolysis of (-)-7): white solid, mp=121°-123° C.

R_(f) (20% ethyl acetate in hexanes)=0.15;

IR: 3285 (br), 3030, 2924, 1496, 1468, 1452, 1402, 1358, 1215, 1134,1103, 1066, 1043, 1022, 904, 738, 694, 661, 630 cm⁻¹ ;

¹ H NMR: δ 7.24-7.37 (m, 20 H), 4.74-4.95 (m, 8 H) 4.42 (dt, J=51, 8.9Hz, 1 H), 3.51-3.78 (m, 4 H), 3.39 (t, J=9.0 Hz, 1 H), 2.49 (d, J=2 Hz,--OH);

Mass spectrum (m/z): 451 (M⁺ --91), 363, 253, 197, 181, 91; exact masscalcd for C₂₇ H₂₈ FO₅ 451.1921, found 451.1920;

Anal. Calcd for C₃₄ H₃₅ FO₅ : C, 75.25; H, 6.50; F, 3.50, found: C,75.19; H, 6.47, F, 3.44;

[α]_(D) ²⁵ =+7.2° (c 1.7, CHCl₃).

(-)-8 (derived by hydrolysis of (+)-7): [α]_(D) ²⁵ =+7.2° (c 1.7,CHCl₃).

Ketone 9: white solid;

IR: 3063, 3030, 2872, 1740, 1496, 1454, 1358, 1213; 1140, 1068, 1026,972, 735, 696 cm⁻¹ ;

¹ H NMR: δ 7.22-7.39 (m, 20 H), 5.04 (ddd, J=49, 9.8, 1.4 Hz, 1 H),4.50-4.92 (m, 8 H), 4.19 (dd, J=9.8, 1.2 Hz, 1 H), 3.86 (t, J=9.2 Hz, 1H), 3.71 (dt, J=13, 9.5 Hz, 1 H), 3.65 (t, J=9.4 Hz, 1 H);

Mass spectrum (m/z): 449 (M⁺ 91), 341, 181, 91, exact mass calcd for C₂₄H₂₆ O₅ F 449.1764, found 449.1763;

Since ketone 9 is not completely homogeneous from NMR analysis, no mp oroptical rotations were measured for either enantiomer.

(+)-10 (derived from (+)-8): Colorless oil;

R_(f) (20% ethyl acetate in hexanes)=0.13;

IR: 3453 (br), 3065, 3032, 2924, 1496, 1454, 1361, 1211, 1153, 1070,1028, 733, 696 cm⁻¹ ;

¹ H NMR: δ 7.27-7.33 (m, 20 H), 4.64-4.90 (m, 8 H), 4.42 (ddd J=44, 9.6,2.5 Hz, 1 H), 4.33 (br s, 1 H), 4.15 (dt, J=11, 9.5 Hz, 1 H), 3.98 (t,J=9.6 Hz, 1 H), 3.41-3.47 (m, 2H), 2.49 (s, --OH);

Mass spectrum (m/z): 451 (M⁺ --91), 253, 197, 181, 91, exact mass calcdfor C₂₇ H₂₈ FO₅, 451.1921, found 451.1920;

Anal. Calcd for C, 75.25; H, 6.50; F, 3.50, found: C, 75.47; H, 6.49; F,3.39;

[α]_(D) ²⁴ =+7.2° (c 2.4, CHCl₃).

(-)-10 (derived from (-)-8): [α]_(D) ²⁴ =+7.2° (c 1.2, CHCl₃).

3-deoxy-3-fluoro-myo-inositol (derived from (+)-10): m.p.=dec. 200° C.;

IR: 3337 (br), 2926, 1107, 1034 cm⁻¹ ;

¹ H NMR (300 MHz, D₂ O): δ 4.43 (DDD, J=47, 9.8, 2.9 Hz, 1 H) 4.27 (dt,J=8.2, 2.9 Hz, 1H), 3.88 (dt, J=11, 9.5 Hz, 1 H), 3.63 (t, J=9.6 Hz, 1H), 3.52 (ddd, J=9.9, 2.6, 1.1 Hz, 1 H), 3.27 (t, J=9.5 Hz, 1 H);

Mass spectrum (m/z): 181 (M⁺ -1), 149, 132, 119, 57, 43;

[α]_(D) ²⁵ =-5.8°±(c 0.38, H₂ O).

EXAMPLE II Synthesis of 3-deoxy-3-fluoro-myo-inositol ##STR6##

Quebrachitol 1, which is extracted from waste-solids of rubber serum, iscommercially available from the Rubber Research Institute of Malaysia.(0.173 g, 0.95 mmol, crystallized from methanol/acetone) was mixed withdiethylaminosulfur trifluoride (DAST, 600 L, 5.75 mmol) at 0° C. and theresulting mixture, after stirring at room temperature under argon for1.5 h, was cooled to -40° C. Methanol (2 mL) was added dropwise(caution). Methanol was evaporated at reduced pressure and a light brownresidue was obtained, which is the fluorinated compound. Such compoundwas dissolved in 32 mL of dry DMF and 2 mL of 2,2-dimethoxypropane alongwith 20 mg of p-TsOH, this step is optional and is utilized to protectthe free hydroxy groups. The mixture was heated under argon at 40° C.for 12 h. Saturated aqueous, sodium bicarbonate solution (10 mL) andwater (50 mL) were added, and the mixture was extracted with 4×50 mlportions of ether. The ethereal extracts were combined, washed withwater and brine, and dried (MgSO₄). Evaporation under reduced pressurefurnished an oily residue which was purified by column chromatography onsilica gel using 20% (v/v) EtOAc/hexane as eluant to givefluorodiacetonide 2 as an oil. Rf 0.25 1:5 EtOAc/hexane. ¹ H NMR(CDCl₃), 300 (MHz) δ 4.6 (br, d, J=47.2 Hz, 1 H), 4.32 (m, 1 H), 4.19(dd, J=7.3, 7.3 Hz, 1 H) 3.98 (ddd, J=31.7, 7.3, 2.6 Hz), 3.7 (br dd,J=19.2, 7.6 Hz), 3.34 (dd, J=0.8, 7.6 Hz), 3.11 (s, 3 H), 1.55 (s, 3 H),1.34 (s, 3 H), 1.22 (s, 3 H), 1.19 (s, 3 H).

The fluorodiacetonide 2 (40 mg. 0.15 mmol) was dissolved in 4 mL ofmethylene chloride and the mixture was cooled to 0° C. Neat borontribromide (200 μL, 12 equiv.) was added via syringe. After stirring for20 h. at room temperature, the mixture was subjected to rotaryevaporation. MeOH (4 mL) was cautiously added at 0° C. and wasevaporated at reduced pressure. The addition and evaporation of MeOH atreduced pressure was carried out several times. Water was added and thebrown aqueous solution was exhaustively washed with CH₂ Cl₂ to removeimpurities. The aqueous phase was evaporated under reduced pressure togive 3-deoxy-3-fluoro-myo-inositol 3 as a pale yellow solid which wascrystallized from MeOH. Yield=26.6 mg, 95%. The product wasspectroscopically identical to the one synthesized in EXAMPLE I.

EXAMPLE III Synthesis of 3-deoxy-3-mercapto-myo-inositol ##STR7##

Quebrachitol diacetonide 1, which is prepared from quebrachitol bytreatment with 2-methoxypropene and an acid, is tosylated and thentreated with BBr₃ to provide the deprotected compound 2, which isconverted to a mixture of bis-acetonides, which are separated by columnchromatography to provide pure 3. Tosylate 3 is reacted sequentiallywith the sodium salt of dimethyldithiocarbamic acid in DMF, then withlithium aluminum hydride and finally with aqueous acid to provide theoptically pure 3-deoxy-3-mercapto-myo-inositol 4.

EXAMPLE IV Synthesis of 3,4-dideoxy-3,4-difluoro-myo-inositol ##STR8##

The 3,4-difluoro isostere of myp-inositol is prepared from the3-fluoro-3-deoxy analog 1. Compound 1 is protected as its bisacetonidederivative, the acetonide mixture separated by chromatography, andcompound 2 having a free C-4 hydroxyl group is treated with DAST. Theisomer 3 of retained stereochemistry is then simply deprotected byaqueous acid treatment to provide the title compound 4.

EXAMPLE V Synthesis of 3,4-dideoxy-3-azido-4-fluoro-myo-inositol##STR9##

The previously described tosylate 1 is reacted with sodium azide in DMFto provide the protected azide 2. Next, DAST treatment of 2 provides thefluorine containing isomer 3 of retained stereochemistry. Cleavage ofthe acetonide protecting groups then affords the desired amino, fluorocontaining isostere 4 of myo-inositol.

EXAMPLE IV Synthesis of 3,4-dideoxy-4-fluoro-mercapto-myo-inositol##STR10##

The previously described tosylate 1 is reacted with the sodium salt ofdimethyldithiocarbamic acid in DMF to give 2. DAST treatment of 2 thenprovides 3 as the major fluorinated product. Lastly, treatment withlithium aluminum hydride and acid catalyzed removal of the acetonidegroups yield the mercapto compound 4.

EXAMPLE VII Synthesis of 2,3-dideoxy-2-amino-3-fluoro-myo-inositol##STR11##

The previously described ketone 1 is subjected to a reductive aminationprocedure to afford the axial amine 2. The benzyl groups are thenremoved by catalytic hydrogenolysis to yield the title compound 3.

EXAMPLE VIII Synthesis of 3,6-dideoxy-3,6-difluoro-myo-inositol##STR12##

The previously described 3-fluoro-3-deoxy isostere 1 is protected as itsbis-acetonide and the mixture of resulting acetonides separated toprovide 2. DAST treatment of 2 provides some of the 6-fluoro product 3of retained stereochemistry. The difluoro isostere 4 is then obtained byaqueous acid treatment of 3.

EXAMPLE IX Synthesis of 3-deoxy-3-cyano-myo-inositol ##STR13##

The previously described tosylate 1 is reacted with sodium cyanide inHMPA to provide the S_(N) 2 displacement product 2. Acetonide removal isbrought about by reaction with aqueous acid to yield the title compound3.

EXAMPLE X Synthesis of 1,3-Dideoxy-1,3-difluoro-myo-inositol ##STR14##

The previously prepared compound 1 is fully protected as 2, thenselectively deprotected to provide 3. The free C-1 hydroxyl group isinverted by an oxidation/reduction sequence, and then transformed to afluoro group by DAST treatment. A final deprotection step then affordsthe desired 1,3-difluoro isostere.

EXAMPLE XI Synthesis of 2,3-dideoxy-2,3-difluoro myo-inositol ##STR15##

The C-1 equatorial hydroxyl group of the previously describedintermediate 1 is protected by benzylation, and the axial C-2 hydroxylgroup is inverted by an oxidation/reduction sequence. DAST treatment ofthis new intermediate 2, followed by deprotection of the hydroxyl groupscompletes the synthesis.

EXAMPLE XII Synthesis of 3,5-dideoxy-3,5-difluoro-myo-inositol ##STR16##

The previously described 3-deoxy-3-fluoro-myo-inositol is protected asits mono-acetonide derivative 1, and 1 is reacted with sodium hydrideand methyl iodide to provide 2 after chromatographic purification. TheC-5 hydroxyl group of 2 is inverted by an oxidation/reduction sequence,and this new alcohol is treated with DAST to yield 3. Lastly,deprotection with boron tribromide is carried out to give this titlecompound 4.

In a fashion identical to that described hereinabove for the preparationof the 1,3-difluoro analogs of myo-inositol, it is possible to replaceany one of the hydroxy groups of 3-deoxy-3-fluoro-myo-inositol by anamino, azido, cyano, mercapto, fluoro, chloro, bromo, iodo or selenolgroup to afford a dideoxyinositol isostere. The strategy requires thatall hydroxy groups except the one to be substituted be protected. Next,the remaining, free hydroxy group is inverted, then activated fordisplacement (e.g., by tosylate or mesylate formation), and adisplacement reaction carried out using the appropriate halogen, carbon,nitrogen, sulfur, or selenium nucleophile. In some situations, inversionof the free alcohol may not be necessary, for the subsequentdisplacement reaction can proceed with retention of stereochemistry(e.g. introduction of a chlorine atom using thionyl chloride). Also, areductive amination process can be used to introduce an amino group intothe protected 3-deoxy-3-fluoro-myo-inositol derivative by oxidation ofits free hydroxyl group to ketone, followed by imine formation andreduction. The desired disubstituted myo-inositol analog is thenobtained by removal of all protecting groups.

In a similar fashion, by starting with any of the mono-substitutedmyo-inositol analogs containing a cyano, mercapto, selenol, fluoro,amino, chloro, bromo, iodo or azido group, and protecting all hydroxylgroups (as well as the non-hydroxyl functional group if necessary)except the one to be substituted, all other dideoxy-disubstitutedanalogs of myo-inositol can be generated. The free hydroxyl group isinverted, activated for displacement, and displaced using theappropriate nucleophile as described hereinabove. In some situations,inversion of the free alcohol group may not be necessary, for thesubsequent displacement reaction can proceed with retention ofstereochemistry. Also, the amino group can be advantageously introducedin certain cases by use of the reductive amination procedure. Lastly,deprotection of all functional groups delivers the requireddideoxy-disubstituted inositol.

5.2 Biological Data EXAMPLE XIII Cell Growth Inhibition Effects Of3-deoxy-3-fluoro-myo-inositol and 1-deoxy-1-fluoro-myo-inositol

The biological effects of the 3-fluoro and 1-fluoro-myo-inositol analogswere tested in PC12 cells, a cell line derived from a ratpheochromocytoma, which has been widely used to study the effects ofvarious growth factors and differentiation promoters as furtherdescribed by Green, L. A., et al., Adv. Cell Neurobiol., 3, 373-414,(1982), the disclosure of which is incorporated herein by reference.

PC12 cells were seeded at a density of 3.3×10⁵ cells/cm² in plasticculture dishes and grown at 37° C. in a humidified atmosphere of 95%air/5% CO₂. The basal medium used in these experiments was RPMI-1640medium (Gibco). In some experiments a specially formulated inositol-freeRPMI-1640 medium (Gibco) was used. This basal medium was supplementedwith either normal or extensively dialyzed horse serum (10%) and fetalcalf serum (5%) (Gibco). Some control dishes were grown in complete RPMImedium, i.e. with myo-inositol and containing non-dialyzed serum(Control 1) and some control dishes were grown in inositol deficientmedium, i.e., RPMI without inositol and with dialyzed serum (Control 2).The inositol analogs were dissolved in distilled H₂ O and added to theculture medium at the indicated concentrations.

Cell counts were performed by removing the medium, washing the cellsgently in PBS with calcium, detaching the cells by 15 minute inducationin PBS/EDTA (0.5 mM) containing 0.1% trypsin, and counting in ahemocytometer.

A dose-response study was performed by growing cells in the presence ofcertain concentrations of the 3-fluoro and the 1-fluoro-myo-inositolanalogs. At the end of five days, the cells were counted by the meansdescribed hereinabove. The results, shown in Table 1, were as follows:

                  TABLE 1                                                         ______________________________________                                        Dose Response for Myo-Inositol Analogs                                        Treatment         Cell Number (×10.sup.5 cells/dish)                    ______________________________________                                        Control Medium                                                                1                 5.1 ± 0.2                                                2                 4.7 ± 0.1                                                1-deoxy-1-fluoroinositol (mM)                                                 0.01              4.53 ± 0.13                                              0.03              4.18 ± 0.07                                              0.10              3.71 ± 0.03                                              0.30              3.29 ± 0.08                                              1.0               2.73 ± 0.13                                              3.0               2.67 ± 0.09                                              10.0              2.68 ± 0.07                                              30.0              2.72 ± 0.03                                              3-deoxy-3-fluoroinositol (mM)                                                 0.01              4.60 ± 0.24                                              0.03              4.49 ± 0.07                                              0.10              4.21 ± 0.05                                              0.30              3.87 ± 0.05                                              1.0               3.21 ± 0.08                                              3.0               2.53 ± 0.04                                              10.0              2.50 ± 0.03                                              30.0              2.36 ± 0.01                                              ______________________________________                                    

Thus, it can be seen that both analogs inhibited cell replication byapproximately 50%, with the 1-deoxy-1-fluoro-myo-inositol analog beingabout five-fold more potent than the 3-deoxy-3-fluoro-myo-inositolanalog.

The time course effect on cell replication was then examined, using theconcentration of 1-fluoro and 3-fluoro myo-inositol analogs determinedto have maximal effects in the dose response study of Table 1. (1 mM and5 mM for the 1-fluoro and 3-fluoro-myo-inositol analogs, respectively).The results, shown in Table 2, were as follows:

                  TABLE 2                                                         ______________________________________                                        Time Course Cell Counts                                                       TREATMENT GROUP                                                                                    1-deoxy-1-  3-deoxy-3-                                          Control Medium 2                                                                            Flouroinositol                                                                            Fluoroinositol                               Time   (Inositol Free)                                                                             (1 mM)      (5 mM)                                       ______________________________________                                        Day 0  .sup. 1.46 × 10.sup.5                                                                 1.46 × 10.sup.5                                                                     1.46 × 10.sup.5                        Day 1   2.52 ± 0.08                                                                             2.10 ± 0.03                                                                            2.12 ± 0.02                               Day 3   8.0 ± 0.3  4.5 ± 0.10                                                                            4.2 ± 0.1                                 Day 5  16.6 ± 1.1 8.4 ± 0.7                                                                              7.6 ± 0.1                                 Day 7  17.0 ± 0.5 10.8 ± 0.6                                                                             10.6 ± 0.6                                ______________________________________                                    

Numbers represent cells per dish, ×10⁵.

Thus, since there was no decline in cell number over time, it isbelieved that the 50% decrease in cell number was the result of apersistent inhibition in cell replication, rather than a cytotoxiceffect resulting in cell death. Furthermore, it is believed that suchinhibition was not the result of simple inositol depletion caused by theblockade of myo-inositol transport into the cells by the myo-inositolanalogs, since control cells grown in inositol-free, dialyzed serum(Control 2), grew almost as well as control cells grown in completemedium, i.e., myo-inositol containing medium, (Control 1), with bothsets of control cultures demonstrating considerably faster replicationthan the fluoro-myo-inositol-treated cultures.

EXAMPLE XIV Effects of Fluoroinositol Analogs on Cell Size Background

As cells grow in culture, the size of an individual cell varies in afairly predictable way. The size of a cell can be expressed as theamount of protein present per cell. Shortly before mitosis (i.e., celldivision), a cell is at its largest diameter and, hence, possesses thegreatest amount of protein per cell. Immediately after mitosis, the cellsize drops considerably, frequently to levels approximately half of thepre-mitotic level.

By investigating the size of a cell, it is possible to determine atwhich point within the cell cycle inhibitors of cell replication areexerting their effects. Accordingly, we investigated the effect of the1- and 3-fluoro-myo-inositol analogs on the size of cells chronicallytreated with such analogs.

Method

Cells were grown and treated with 1- and 3-fluoro-myo-inositol analogsas described in Example XIII. On the days indicated, cells were gentlyharvested without disruption, and the number of cells counted intriplicate in a cell counting chamber. Following determination of thenumber of cells per sample, the remainder of the cells were pelleted ina microcentrifuge as described hereinabove, and the protein in thesample was determined by the method of Lowry. Thus, it was possible toderive from these two data sets the amount of protein per cell, which isexpressed as nanograms of protein per cell, as indicated in Table 3:

                  TABLE 3                                                         ______________________________________                                        Effects of Fluoroinositol Treatment on Cell Size                              Treatment                         3-deoxy-3                                   Duration          1-deoxy-1-Fluoroinositol                                                                      Fluoro-                                     (Days) Control 2  (nanograms protein/cell)                                                                      inositol                                    ______________________________________                                        1      0.110 ± 0.01                                                                          0.082 ± 0.01 0.086 ± 0.01                             3      0.118 ± 0.03                                                                          0.135 ± 0.01 0.259 ± 0.01                             5      0.181 ± 0.03                                                                          0.160 ± 0.01 0.320 ± 0.01                             7      0.063 ± 0.02                                                                          0.168 ± 0.02 0.343 ± 0.03                             ______________________________________                                    

Results

As can be seen in Table 3, control cells demonstrated, over time, theexpected progressive increase in the average amount of protein per cell.Thus, the protein per cell on day one was approximately 0.1 nanogramsper cell and this had almost doubled (0.18 nanograms/cell) by day five.Between day 5 and day 7, the majority of cells in the control sampledivided and the average protein per cell had dropped to approximately0.06 nanograms protein per cell, reflecting the smallest, post-mitoticcell size. This pattern is consistent with the reported doubling timefor this cell line.

In contrast to this normal pattern, the 1-fluoro-myo-inositol treatedcells showed an arrest in their cell cycle. Cellular size on day one wasapproximately the same as that of controls (as expected) and the amountof protein per cell steadily increased to a plateau approximately twicethat of the protein levels on day one. Thus, cells treated with1-fluoro-myo-inositol were able to continue to increase in size up tothe point at which they should receive a signal to divide. However, thissignal was apparently lacking and cells arrested in a pre-mitotic state(termed late G2), as reflected by their size, which was approximately2-fold greater than controls.

In contrast to both the control and 1-fluoro-myo-inositol treated cells,the 3-fluoro-myo-inositol treated cells demonstrated a most remarkableand unique pattern of cell growth. These cells likewise began withnormal levels of cellular protein reflecting a normal size. However,these cells continued to increase steadily in size without undergoingcell division. Thus, by day seven, 3-fluoro-myo-inositol treated cellshad increased to a size about 3.5 times as large as control cells.Nevertheless, the rate of cell replication had slowed considerably, asindicated by EXAMPLE XII. Thus, it seems that the 3-fluoro-myo-inositolcompound disrupts the ability of the cell to initiate mitosis (i.e.,cell division), but that cell size continues to increase. This indicatesfundamental desynchronization of the internal signaling systems of thecell with regard to control of cell replication and cell division, whichinvolve the conjoint regulation of cell size and cell replication.

EXAMPLE XV Effects of 1-deoxy-1-Fluoro and 3-deoxy-3-Fluoro-Myo-inositolAnalog on Inositol Transport Background

To determine if the effect of the fluoro-myo-inositol analogs might bemediated by the blockade of the myo-inositol transporter, which isresponsible for uptake and accumulation of myo-inositol into the cell,the ability of these two compounds to block [³ H]myo-inositol transportwas studied directly.

Method

The uptake of [³ H]myo-inositol by the myo-inositol transporter wasdetermined as follows:

PC12 cells were gently detached and maintained in a suspension culturein RPMI medium (both myo-inositol and serum free) at 37° C. [³H]myo-inositol was added to this culture at a concentration of 1μCi/ml.Aliquots of this suspension were removed at the indicated time, dilutedin 10 volumes of ice-cold PBS containing 10 mM unlabelled myo-inositolto stop the uptake process, centrifuged at 4° C. for 20 minutes at 50xg.The cell pellet, which contained the transported [³ H]myo-inositol, wasdissolved in dilute NaOH and the radioactivity quantified by liquidscintillation counting. The results, shown in Table 4, were as follows:

                  TABLE 4                                                         ______________________________________                                        Effects of 1-deoxy-1-Fluoro and 3-deoxy-3-Fluoro                              Myo-Inositol Analogs on Inositol Transport                                                    Affinity                                                                      Constant Maximal Transport Rate                               Compound        K(μM) Vmax (nMol/mg/hr)                                    ______________________________________                                        Myo-inositol      45     2.27                                                 1-Fluoro-myo-inositol analog                                                                  1,430    2.27                                                 3-Fluoro-myo-inositol analog                                                                    250    2.27                                                 ______________________________________                                         The Ka and Vmax of myoinositol were determined from LineweaverBurke plots     of the uptake of [.sup.3 H]myoinositol (25 nM) in the presence of             increasing concentrations of unlabeled myoinositol in PC12 cells. The Ki      of the two fluoromyo-inositol analogs was determined by conducting the        uptake studies in the presence of a fixed concentration (1 mM) of each        myoinositol analog.                                                      

These results indicate that the 1-fluoro and 3-fluoro-myo-inositolanalogs did block [³ H]myo-inositol uptake, but with a reversed potencycompared to their effects on cellular replication. Indeed, the 3-fluoromyo-inositol analog was 5.7 times as potent as the 1-fluoro myo-inositolanalog in blocking myo-inositol transport. These results demonstratedthat the growth inhibitory effects of 1-fluoro and 3-fluoro myo-inositolanalogs accrued distal to the transport site for myo-inositol, sincethere was a reversed selectivity for these two analogs with regard totheir growth and transport inhibitory effects.

EXAMPLE XVI Effects of Fluoroinositol Analogs on Inositol Incorporationinto Cell Plasma Membranes Background

The results described hereinabove demonstrate that 1-deoxy-1-fluoro and3-deoxy-3-fluoro-myo-inositol analog are capable of inhibiting cellularreplication in a dose dependent manner. Furthermore, the mechanism ofthis inhibition involves the intracellular myo-inositol signallingpathways since the 1-fluoro-myo-inositol analog was 5-fold more potentthan the 3-fluoro-myo-inositol analog at inhibiting cell replication,but had an affinity for the myo-inositol transporter that was 5.7- foldlower than the 3-fluoro-myo-inositol analog.

Since myo-inositol must be incorporated into the cellular plasmamembranes in order for it to serve its role as a second messenger, wetherefore verified the ability of the 1- and 3-fluoro-myo-inositolanalogs to perturb the incorporation of myo-inositol into cellularplasma membranes by the enzyme CDP-diacylglycerol-inositolphosphatidyltransferase (phosphatidylinositol synthetase).

We predicted that cells exposed to the 1-fluoro-myo-inositol analogwould be unable to incorporate any myo-inositol into their cellularplasma membranes, since the 1 position necessary for themyo-inositol-diacylglycerol linkage was blocked by the fluorine at thisposition. In contrast, we predicted that the 3-fluoro-myo-inositolcompound would be incorporated into the cellular plasma membranes sincethe 1 position was free.

These tests were conducted by treating cells for either 1, 3, 5, or 7days with the myo-inositol analogs, followed by a brief exposure toradiolabeled myo-inositol ([³ H]myo-inositol). The extent ofincorporation of tritiated myo-inositol would, therefore, reflect theextent to which phosphatidylinositol and its phosphorylated analogs aredeficient in the cellular plasma membranes.

Methods

Cells were grown as described hereinabove. Twenty-four hours afterplating, the medium was changed and cells were exposed to eitherinositol free medium (Control 2) or medium containing either the 1- orthe 3-fluoro-myo-inositol analog. At four different time points--1, 3,5, and 7 days--cells were tested for their ability to incorporate radiolabeled [³ H]myo-inositol. This test was conducted by removing themedium and replacing it with RPMI medium (both myo-inositol and serumfree) containing only [³ H]myo-inositol The cells were exposed to thismedium for a period of one hour, following which they were washed threetimes with phosphate-buffered saline containing excess unlabeledmyo-inositol (10 mM). Cells were lysed by treatment with trichloroaceticacid (TCA) and sonicated to insure complete disruption. The cellularhomogenate was then collected and spun in a microfuge tube at 12,000r.p.m. for 15 min. Samples from both the pellet and supernatant werethen obtained and the levels of radioactivity in each were determined.Radioactivity in the supernatant corresponded to the amount of free [³H]myo-inositol in the cytoplasm of the cell, whereas the radioactivitypresent in the TCA-precipated pellet represented [³ H]myo-inositolincorporated into the cellular plasma membranes.

Results

The results, shown in Table 5, were as follows:

                                      TABLE 5                                     __________________________________________________________________________    Uptake and Incorporation of [.sup.3 H]myo-inositol into PC12                  Cells Following Treatment with Inositol Analogs                                               1-deoxy-    3-deoxy-                                          Control         1-Fluoro-Ins                                                                              3-Fluoro-Ins                                      Day Free  Bound Free  Bound Free  Bound                                       __________________________________________________________________________    1   8840 ± 146                                                                       367 ± 15                                                                         8365 ± 787                                                                       392 ± 16                                                                         3326 ± 253                                                                       354 ± 10                                 3   3749 ± 776                                                                       794 ± 128                                                                        4751 ± 291                                                                       4614 ± 141                                                                       2929 ± 189                                                                       761 ± 42                                 5   3967 ± 449                                                                       759 ± 119                                                                        4295 ± 627                                                                       2143 ± 314                                                                       2900 ± 383                                                                       414 ± 52                                 7   1942 ± 484                                                                       397 ± 100                                                                        7569 ± 757                                                                       3726 ± 588                                                                       4095 ± 161                                                                        676 ± 113                               __________________________________________________________________________     Cells were treated for the indicated time periods with the inositol           analogs, and then exposed for one hour to [.sup.3 H]myoinositol. Numbers      represent DPM/10.sup.6 cells.                                            

As can be seen, this procedure allowed for the uptake of a significantamount of radiolabeled myo-inositol. Control cells demonstrated a robustuptake of radiolabeled myo-inositol into the cytoplasm on all daysexamined, but only a relatively small fraction of the total radioactivemyo-inositol (ranging from 4% on day one to 17% on day seven) wasactually incorporated into the cellular plasma membranes after beingtransported into the cell. This is consistent with our understandingthat myo-inositol is of vital importance for the maintenance of cellcycle control and that the majority of sites available for linkage tomyo-inositol in the cellular plasma membranes are occupied bymyo-inositol at all times.

In contrast, cells treated with the 1-fluoro-myo-inositol analogdemonstrated a very different pattern of incorporation. On day one,these cells were not remarkably different from control cells in eitherthe amount of free or incorporated [³ H]myo-inositol. However, thesecells rapidly became myo-inositol deficient as evidenced by their markedincorporation of radiolabeled myo-inositol into the cellular plasmamembranes on days 3, 5, and 7. Thus, after only several days of exposureto the 1-fluoro-myo-inositol analog, there was a 10-fold increase in thenumber of unoccupied sites for myo-inositol in the cellular plasmamembranes. This is consistent with the ability of 1-fluoro-myo-inositolanalog to compete for myo-inositol uptake, but once inside the cell, tobe an inactive substrate for the key enzyme phosphatidylinositolsynthetase (CDP-diacylglycerol-inositol phosphatidyltransferase;E.C.2.7. 8.11), which catalyses the linkage of myo-inositol tophosphatidic acid, which is present in the cellular plasma membranes.

The effects of the 3-fluoro-myo-inositol analog were also consistentwith our predictions. Although the cells continued to be able toaccumulate myo-inositol without difficulty, the level ofradioactively-labeled myo-inositol that was incorporated into thecellular plasma membranes was quite low. This indicates that the3-fluoro-myo-inositol analog did serve as a substrate for inositolsynthetase and that the membrane sites for myo-inositol were indeedoccupied by the 3-fluoro-myo-inositol analog.

Accordingly, the 1- and the 3-fluoro-myo-inositol analogs would beexpected to disrupt the phosphatidylinositol cycle at different points.Furthermore, 1-fluoro-myo-inositol should disrupt myo-inositol signalingcompletely, whereas the 3-fluoro-myo-inositol analog compound shoulddisrupt signaling mediated by myo-inositol compounds phosphorylated atthe 3 position.

These results further provide conclusive evidence that both the 1-fluoroand 3-fluoro myo-inositol analogs serve as inhibitors of cell growthbased upon their ability to interfere with phosphatidylinositol cycle.Accordingly, these fluoro-myo-inositol analogs should have utility in avariety of settings in which the transmission of signals via thephosphatidylinositol pathway is of importance. For example, thesecompounds should be useful in the study of cell growth, both in vivo andin vitro, as well as in the treatment of disorders in which cellularproliferation is disturbed. However, the phosphatidylinositol cycle isof fundamental importance for a number of physiological processesunrelated to cell replication, so that these compounds should findutility in other areas as well. By way of example, the activation ofmany neurotransmitter receptors is known to result in the stimulation ofthe PI cycle. Accordingly, the study of neural function as it relates tothe PI cycle should be facilitated by the use of these compounds.Furthermore, these compounds should find utility in the treatment ofdisorders in which excessive neurotransmitter activity has beenimplicated. For example, lithium salts have been used for many years inthe treatment of bipolar mood disorder, and it is now known that lithiumblocks the PI cycle, as do the inositol analogs described hereinabove.Accordingly, these compounds should find utility in the treatment ofbipolar disorder. Inositol signaling pathways are also known to beimportant in the proper functioning of important blood elements (e.g.,lymphocytes, leukocytes, platelets) and the analogs describedhereinabove should be useful in the study of these systems as well asthe treatment of certain disorders of these systems. The examples givenabove are incorporated solely for the purpose of illustrating the knownimportance of the PI cycle, and the utility to be derived from compoundscapable of inhibiting the PI cycle. Therefore, the utility of thesecompounds should not be construed as being limited to the few examplesdescribed hereinabove.

What is claimed is:
 1. A compound represented by the general formula II:##STR17## wherein: R¹, R², R³, R⁴, R⁵ and R⁶ is each independentlyselected from the group consisting of OH, F, N₃, NH₂, SH, SeH, Cl Br, Iand CN; with the proviso:(i) four or five of said R¹, R², R³, R⁴, R⁵ andR⁶ are OH; (ii) if one of said R¹, R², R³, R⁴, R⁵ and R⁶ is N₃, then oneof said R¹, R², R³, R⁴, R⁵ and R⁶ is selected from the group consistingof F, NH₂, SH, SeH, Cl, Br, I and CN; (iii) if one of said R¹, R¹ 2, R³,R⁴, R⁵ and R⁶ is NH₂, then one of said R¹, R², R³, R⁴, R⁵ and R⁶ isselected from the group consisting of F, N₃, SH, SeH, Cl, Br, I and CN;and (iv) if said R⁴, R⁵ or R⁶ is F, Cl, Br or I then four of said R¹,R², R³, R⁴, R⁵ and R⁶ are OH.
 2. A compound according to claim 1 whereinfive of said R¹, R², R³, R⁴, R⁵ and R⁶ are OH.
 3. The compound of claim1 wherein one of said R¹, R², R³, R⁴, R⁵ and R⁶ is F or SH.
 4. Thecompound method of claim 3 wherein one of said R¹, R², R³, R⁴, R⁵ and R⁶is F.
 5. The compound of claim 1 wherein R¹ is selected from the groupconsisting of F, SH, SeH, Cl, Br, I and CN.
 6. The compound of claim 5wherein R¹ is F or SH.
 7. The compound of claim 6 wherein R¹ is F. 8.The compound of claim 1 wherein R³ is selected from the group consistingof F, SH, SeH, Cl, Br, I and CN.
 9. The compound of claim 8 wherein R³is F or SH.
 10. The compound of claim 9 wherein R³ is F.
 11. Thecompound of claim 1 wherein four of said R¹, R², R³, R⁴, R⁵ and R⁶ areOH.
 12. The compound of claim 11 wherein two of said R¹, R⁴ and R⁵ areindependently selected from the group consisting of F, N₃, NH₂, SH, SeH,Cl, Br, I and CN.
 13. The compound of claim 12 wherein said R⁴ and R⁵are independently selected from the group consisting of F, N₃, NH₂, SH,SeH, Cl, Br I and CN.
 14. The compound of claim 11 wherein two of saidR¹, R², R³, R⁴, R⁵ and R⁶ are selected from the group consisting of Fand SH; F and NH₂ ; NH₂ and SH; SH and SH; and F and F.
 15. The compoundof claim 14 wherein two of said R¹, R², R³, R⁴, R⁵ and R⁶ are F and F.16. A pharmaceutical composition comprising a therapeutically effectiveamount for inhibiting the phosphatidylinositol cycle in a manual of acompound represented by the general formula III: ##STR18## wherein: R¹,R², R³, R⁴, R⁵, and R⁶ is each independently selected from the groupconsisting of OH, F, N₃, NH₂, SH, SeH, Cl, Br, I and CN; with theproviso:(i) four or five of said R¹, R², R³, R⁴, R⁵, and R⁶ are OH; (ii)if one of said R¹, R², R³, R⁴, R⁵ and R⁶ is N₃, then one of said R¹, R²,R³, R⁴, R⁵, and R⁶ is selected from the group consisting of F, NH₂, SH,SeH, Cl, Br, I and CN; (iii) if one of said R¹, R², R³, R⁴, R⁵, and R⁶is NH₂, then one of said R¹, R², R³, R⁴, R⁵, and R⁶ is selected from thegroup consisting of F, N₃, SH, SeH, Cl, Br, I and CN; and (iv) if saidR⁴, R⁵, and R⁶ is F, Cl, Br, or I then four of said R¹, R², R³, R⁴, R⁵,and R⁶ are OH, and a pharmaceutically acceptable carrier.